Correlation of axillary osmidrosis to a SNP in the ABCC11 gene determined by the Smart Amplification Process (SmartAmp) method

Journal of Plastic, Reconstructive & Aesthetic Surgery (2010) 63, 1369e1374

Correlation of axillary osmidrosis to a SNP in the ABCC11 gene determined by the Smart Amplification Process (SmartAmp) method* Y. Inoue a, T. Mori b, Y. Toyoda c, A. Sakurai c,d, T. Ishikawa c,d, Y. Mitani d,e, Y. Hayashizaki d, Y. Yoshimura a, H. Kurahashi b, Y. Sakai a,* a

Department of Plastic and Reconstructive Surgery, Toyoake, Japan Division of Molecular Genetics, ICMS, Fujita Health University, Toyoake, Japan c Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan d Omics Sciences Center (OSC), RIKEN Yokohama Institute, Yokohama, Japan e K.K. Dnaform, Yokohama, Japan b

Received 7 November 2008; accepted 18 June 2009

KEYWORDS Axillary osmidrosis; Earwax type; Single nucleotide polymorphism (SNP); ATP-binding cassette (ABC) transporter; ABCC11; Smart Amplification Process (SmartAmp)

Summary Axillary osmidrosis (AO) is caused by apocrine glands secretions that are converted to odouriferous compounds by bacteria. A potential link between AO and wet earwax type has been implicated by phenotype-based analysis. Recently, a non-synonymous single nucleotide polymorphism (SNP) 538G> A (Gly180Arg) in the human adenosine triphosphate (ATP)-binding cassette (ABC) transporter ABCC11 gene was found to determine the type of earwax. In this context, we examined a relationship between the degree of AO and the ABCC11 genotype. We have genotyped the SNP 538G> A in a total of 82 Japanese individuals (68 volunteers and 14 AO patients) by both DNA sequencing and the recently developed Smart Amplification Process (SmartAmp). The degree of AO in Japanese subjects was associated with the genotype of the ABCC11 gene as well as wet earwax type. In most AO patients investigated in this study, the G/G and G/A genotypes well correlated with the degree of AO, whereas A/A did not. The specific SmartAmp assays developed for this study provided genotypes within 30 min directly from blood samples. In East Asian countries, AO is rather infrequent. Although the judgement of the degree of AO prevalence is subjective, the SNP 538G> A in ABCC11 is a good genetic biomarker for screening for AO. The SmartAmp method-based genotyping of the ABCC11 gene would provide an accurate and practical tool for guidance of appropriate treatment and psychological management for patients. ª 2009 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

* Presented at the 16th Research Council Meeting of Japan Society of Plastic and Reconstructive Surgery, in Kobe, Hyogo, Japan, 11 and 12 October 2007. * Corresponding author. Tel./fax: þ81 562 93 9249. E-mail address: [email protected] (Y. Sakai).

1748-6815/$ - see front matter ª 2009 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bjps.2009.06.029

1370 Axillary osmidrosis (AO) or bromhidrosis is a physical condition that is often perceived, especially by younger women, as a distressing and troublesome problem. In fact, the condition of AO has been treated and recognised as a disease that is covered by the national health insurance system in Japan. Sweat produced by axillary glands, including apocrine, eccrine, apoeccrine and sebaceous glands, is odourless.1 Secretions from apocrine glands can be converted to compounds by bacteria (Corynebacteria), which have an unpleasant smell associated with body odour.1,2 The mechanism of producing the major odour-causing compounds in the human axilla is still a subject of controversy. Because ancillary odour first appears at puberty when apocrine glands become fully developed, early studies were first focussed on volatile odouriferous steroids.3,4 More recently branched and unsaturated fatty acids as for example (E )-3-methyl-2-hexenoic acid (3M2H) and 3-methyl-3-hydroxylhexenoic acid (HMHA), have been identified as the key components of odor rather than steroids.5 Released from apocrine secretions, 3M2H is carried to the skin surface bound to apolipoprotein D (also known as apocrine secretion odour-binding protein 2),5 and is also produced from 3M2H-Gln by bacterial N-acylaminoacylase in the axilla.6 In addition, volatile sulphur compounds, such as 3-methyl-3-sulphanylhexan-1-ol, have been identified as another source of typical sweat/onion-like smells.7,8 In East Asian countries, AO is rather infrequent; hence, the social norm is generally a natural expectation for axillary odourlessness. Histological findings reveal significantly numerous and larger-sized apocrine glands in osmidrosis patients, compared with the controls, suggesting the existence of two different phenotypes.9,10 Furthermore, wet earwax has been shown to be always inherited as a Mendelian dominant trait, and accompanied with osmidrosis.9,11,12 Human earwax is a secretory product of ceruminous apocrine glands and shown to be either wet or dry in consistency. Dry earwax is common in East Asians, whereas wet earwax is common in most all other populations. In 2006, a single nucleotide polymorphism (SNP) in the ABCC11 gene, at nucleotide 538 (538G> A; 180Gly> Arg) in exon 4, was identified as a determinant for either wet or dry types of earwax.13 The AA genotype was linked to dry earwax, whereas GA and GG were connected to the wet type. Moreover, a deleted mutation of 27 base pairs (bps) in exon 29 (D27) in the G allele shows an equivalent effect to the A allele on ABCC11. Both the SNP (538G> A) and the deletion (D27) are believed to cause an inactive form of the ABCC11 product.14 ABCC11 is a novel ABC transporter identified by database search, and cloned from a cDNA library of human adult liver.15e17 Known as multidrug-resistance-associated protein 8 (MRP8), the ABCC11 gene product contains two ATP-binding cassettes (ABC) and 12 transmembrane helices. Human ABCC11 and ABCC12 gene are located on human chromosome 16q12.1 in a tail-to-head orientation with a separation distance of about 20 kb.17 The predicted amino acid sequences of both gene products show a high similarity to those of ABCC4 and ABCC5.18 However, there is no putative mouse or rat orthologous gene corresponding to human ABCC11.19 This fact indicates that ABCC11 is not an orthologous gene but rather a paralogous gene generated by gene duplication in the human genome.

Y. Inoue et al. In this study, we examined the relationship between the degree of AO and the genotype of ABCC11. Our results support that a condition of AO in Asians can be recognised by genetic diagnosis, which would be conclusive for providing objective evidence to authorise a treatment policy. Procedures for the diagnosis and the practical management of patients with AO will be facilitated by the new method described herein. The SmartAmp method is a unique genotyping technology that can detect a genetic mutation in about 30 min under isothermal conditions and in a single step by using a drop of whole blood.20

Patients and methods All the procedures were performed according to the protocol approved by the Ethical Review Board of Fujita Health University School of Medicine. Eighty-two individuals (68 volunteers and 14 patients) participated in this study after giving written informed consent. All examinees expressed their subjective views on the symptoms of AO by answering survey questions concerning themselves and family members (Table 1). An impartial practitioner, Y.I., who is without olfactory impairment and a specialist in plastic surgery, conducted all the examinations for diagnosis of the study participants. The olfactory function of Y.I. was confirmed by an intravenous olfactory test (Alinamin Test) and an electroolfactogram. To test the degree of odour, gauze inserted in the axilla for 3 min was estimated by its smell. Earwax type was determined using a cotton swab stirred in the external ear canal. Another researcher, who was blind to the examinees’ physical background, carried out DNA sequencing to determine the genotype of each study participant. Statistical analysis on the relationship between two variables was evaluated by Fisher’s exact test. Individual genomic DNA was obtained from 2 ml of whole blood using QuickGene-610 L (Fujifilm Co., Tokyo, Japan) according to the manufacturer’s protocol. Sequence data for the human genome was obtained from the National Center for Biotechnology Information (NCBI) database. To sequence the SNP (538G> A), the following PCR primers were designed: P1: 50 -TGT CACATGCAAAGAGATTCC-30 and P2: 50 -CTCCTGGCATGGACTTGAACA30 . To identify the D27 mutation, we also designed the set of primers: P3: 50 -AG GTCTCTAGGGCCTGAAGTA-30 and P4: 50 AGCCTTCACCTTCC CATTGCC-30 . All sequences (538G> A and D27) were performed using 3100 Genetic Analyzer (Applied Biosystems Ltd., Tokyo, Japan) according to the manufacturer’s protocol. Table 1

Subjective findings on the examinees Earwax

AO

Family history of AO

Yes No a

Dry 49

1

b

Volunteers Wet 19a 12b (n Z 68) Patients (n Z 14)

Dry 1a Wet 13a

1 13

Yes

No

Unclear

48 1 7b 9

43 7

5 3

0 0 0 9

1 3

0 1

b

AO; axillary osmidrosis. a p < 8.2 x 106. b p < 7.9 x 108 (Fisher’s exact test).

Axillary osmidrosis, ABCC11 and SmartAmp

1371

Figure 1 Three different patterns in the SNP (538G> A) of ABCC11. Wave patterns of DNA sequence analysis (upper panel) and the corresponding results of SmartAmp genotyping (lower panel). The arrows indicate at nucleotide 538 in ABCC11. Amplification time course of SmartAmp shows three diploid genotypes. dR; baseline-subtracted fluorescence reading.

We developed SmartAmp assays for the SNP in ABCC11, which was described in our recent paper.14 Basic mechanisms of the method and primer design are introduced in detail on the website (http://www.dnaform.jp/smartamp/ index_e.html). Briefly, a drop of whole blood obtained from each individual was denatured at 98  C for 3 min, and mixed with reaction buffer containing SYBR Green I (Takara Bio Inc., Otsu, Japan) and Aac DNA polymerase (K. K. Dnaform, Yokohama, Japan). A real-time PCR reaction was performed at 60  C for 30 min. The fluorescence intensity of SYBR Green I dye was monitored with Mx3000P system (Agilent Technologies, Santa Clara, CA, USA).

Results Sixty-eight volunteers (30 males and 38 females; age range 21e55 years; average age 29.1 years) and 14 patients (one male and 13 females; age range 19e65 years; average age 36 years) were examined.

dry group, compared with 12 (12/19, 63.1%) in the wet group (p < 7.9  108).

ABCC11 locus sequenced by DNA sequencer and SmartAmp We performed sequencing analysis for determination of both the SNP (538G> A) and the deletion (D27). The expected sizes of PCR products obtained by using the P1/P2 primer pairs for exon 4 (285 bp) and the P3/P4 primer pairs for exon 29 (226 bp) were obtained. Sequencing results of the PCR products matched previously published data for the ABCC11 gene locus.13 All three possible genotypes at nucleotide position 538, representing the wild-type homozygote (GG), heterozygote (GA) and mutant homozygote (AA), could be observed by sequencing and SmartAmp (Figure 1). Notably, no individual in this study showed the D27 mutation (data not shown).

Analysis on subjective findings

Comparison and analysis of subjective findings, diagnosis and sequencing results

Subjective findings revealed a strong association among earwax types, subjective symptoms of osmidrosis and familial tendency (Table 1). Accordingly, we divided the study group into two groups: the dry group and the wet group. First, we compared a distribution of earwax types between the volunteers and the patients. The percentage of wet earwax in the patients (13/14, 92.9%) was significantly higher than that (19/68, 27.4%) in the volunteers (p < 8.2  106). Second, we determined subjective symptoms of osmidrosis between two earwax types in the volunteers. AO was present in only one (1/49, 2.0%) in the

To correlate phenotype and genotype data, we compared subjective findings and diagnosis with the sequencing data (Figure 2). Seven of 82 individuals (indicated as red squares in Figure 2) showed a discrepancy between subjective findings and diagnosis. After meticulous examinations by the physician, six people except one (indicated as an asterisk in Figure 2) agreed to be simply wrong in their personal assessments. The patient who persisted in her personal assessment of osmidrosis, despite objective data, was considered to have osmophobia; an anxiety disease delineated by paranoia of personal body odour. Concerning

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Figure 2 Summary of subjective findings, diagnosis and genotyping. Red squares in numbers indicate the examinees with a discrepancy between their personal assessments and diagnosis by the physician. Blue squares in numbers indicate the individuals who were judged dry earwax type and without AO. A person indicated as asterisk did not agree with the diagnosis. AO; axillary osmidrosis.

a degree of odour, the diagnosis contained a subjective tendency, but at least examinees with the dry earwax type did not possess AO. The sequencing data in this study totally supported the diagnosis on earwax types (Table 2 and Figure 2). The percentage of AO individuals with wet earwax (26/30, 86.7%) was significantly higher than those (0/52, 0%) with dry earwax (p < 1.7  1017). Earwax types obtained by the diagnosis were perfectly matched to genotypes (p < 4.5  1023). Collectively, people with AA genotype correlated with the dry earwax and did not show AO (indicated as blue squares in Figure 2).

Discussion In the present study, we have demonstrated a significant relationship between AO and anSNP of the ABC transporter ABCC11 gene. Moreover, the results obtained by traditional DNA sequence analysis were completely consistent with those by the SmartAmp method, suggesting that the SmartAmp method can be an alternative tool for screening of AO in Asians or other populations.

Strategy for diagnosis of AO Diagnosis for AO consists of a physical evaluation and consideration of subjective and objective findings. Osmidrosis and a simple sweaty condition are sometimes confused by the general population. We accepted the criterion that AO is caused by secretions from apocrine glands and characterised by acid odour. However, a tool for measuring an objective degree of odour has not been developed, which makes reliability of the diagnosis uncertain. Often

employed is a starch iodine test to outline the area of excessive sweating.21 A skin biopsy in the axilla is also useful for detecting the histological difference of apocrine glands between AO patients and controls.10 In 1937, Adachi first reported a relationship between the wet earwax type and AO.9 Based on empirical observation, wet earwax was found to be a dominant phenotype intimately related with osmidrosis.11,12 Indeed, Ou et al. indicated that all of AO patients (20/20) had the wet earwax type.22 Recently, Yoo et al. have also showed that 96% (860 out of 896) of AO patients possessed wet earwax symptoms, and they proposed the algorithm for the diagnosis of AO focusing on the wet earwax type and family history.12 In the present study, AO was observed in the groups of G/G homozygote and G/A heterozygote in the ABCC11 gene, whereas AO was never observed in the group of AA homozygote. The observations rationalise the validity of genotyping the ABCC11 gene in a clinical setting screening for AO among the patients (Table 2, Figure 2).

Table 2

Diagnosis and the SNP in ABCC11 Earwax

AO Yes

Individuals (n Z 82)

Dry 52 Wet 30

a

0 26a

Genotyping No a

52 4a

GG 0 1

AO; axillary osmidrosis. a p < 1.7 x 1017. b p < 4.5 x 1023 (Fisher’s exact test).

GA 0 29

(GG þ GA) b

(0 ) (30b)

AA 52b 0b

Axillary osmidrosis, ABCC11 and SmartAmp

Figure 3

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Algorithm for diagnosis and treatments of AO.

Algorithm for diagnosis and treatment of AO There have been various methods for the treatment of AO. Non-surgical methods include antiperspirants, pharmacotherapy (e.g., anticholinergics, beta-blockers, benzodiazepines) and Botulinum toxic type A (Botox), which are employed basically to prevent hyperhidrosis and only have temporary effects.21 To treat palmar and axillary hyperhidrosis, thoracoscopic sympathicotomy has been employed, but compensatory sweating at a higher rate still bothers the patients.23 A purpose of surgeries in the axilla is to remove most of apocrine glands. The methods are classified into two types: one is en bloc resection including the skin, and the other is resection of subcutaneous tissue including sweat glands. Radical resection (en bloc resection) of the axillary skin can remove a whole structure of the odiferous origin but can cause scar contracture that prevents range of movement of the shoulder joint.24 New surgical approaches have been tried to focus on preserving the overlying skin.12,25e28 The procedures, however, are associated with various complications, such as skin necrosis, scar contracture, haematoma, seroma and unfavourable scarring. Thus, surgical treatments are effective for AO but are often not a comfortable solution for either the patients or the surgeons. Based on the results, we hypothesised that people with AA genotype were not directly connected to AO (Table 2; Figure 3). There are people who display an excessive fear, aversion or psychological hypersensitivity to smells or odours.29 They tend to opt for aggressive surgical treatments and are sometimes categorised as having osmophobia. We believe objective evidence can help prevent unnecessary treatments on such patients.

Relationship between axillary odour and ABCC11 Transcript analyses have shown that human ABCC11 mRNA is ubiquitously expressed in adult and foetal tissues.16,17 ABCC11 was shown to transport a variety of lipophilic anions, including cyclic nucleotides, glutathione conjugates such as leukotriene C4 (LTC4) and S- (2,4-dinitrophenyl)glutathione (GS-DNP), steroid sulphates such as estrone 3-sulphate (E1S) and dehydroepiandrosterone 3-sulphate (DHAES), glucuronides such as estradiol 17-b-D-glucuronide

(E217bG), monoanionic bile acids glycocholate and taurocholate and folic acid and its analogue methotrexate (MTX). Among them, DHEAS is considered as a good substrate for the wild type (Gly180) of ABCC11 because of its high affinity.30,31 Furthermore, DHEAS was detected in the extract of axillary hairs.32 On the other hand, as we have recently reported, the SNP (538G> A; Arg180) variant of ABCC11 lacks the transport activity, since this variant protein is improperly folded in the endoplasmic reticulum and rapidly degraded in proteasomes.14 Based on this study, we conclude that the SNP of 538G> A in the ABCC11 gene can provide scientific evidence that is equivalent to the objective assessment rendered by an experienced clinician. Genetic data can provide a tool for psychological management of patients believed to be suffering from osmophobia, to guide them towards appropriate treatment regimens of a less-invasive nature. Furthermore, we here show evidence that the SmartAmp can provide a simple and accurate alternative for genotypic assessment of individuals being evaluated for osmidrosis, and such objective data can be effective for accurate diagnosis even by inexperienced clinicians. The method enables clinicians to detect the SNP in 30 min by using one drop (1e5 ml) of whole blood from a patient. We think the SmartAmp method should be widely used in the genetic testing of AO patients.

Acknowlegements We thank K. Yoshiura, A. Lezhava, K. Miyazaki, A. Sakakibara, T. Gomi, Y. Inaba and S. Imamura for their useful suggestions and cooperation. We also wish to thank K. Takami for his help in collecting samples and for technical assistance. Y.T. is a JSPS fellow. This work was partially supported by research grants from Fujita Health University School of Medicine and RIKEN Omics Science Center from MEXT as well as by the Japan Science and Technology Agency (JST) research project Development of the world’s fastest SNP detection system.

Conflict of interest The authors declare that they have no conflicts of interest.

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